Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.


Supporting Information
Table S2.Identification of the five N-glycans located within glycopeptides at Asn88, Asn125, Asn174, and Asn217 on the long chain and at Asn537 on the short chain of HSulf-2.
Table S3.List of the most abundant glycopeptides containing Asn residue issued from trypsin and PNGase F treatment and detected after CID/ETD analysis.Each site of glycosylation was confirmed by the detection of deamidation introduced by PNGase F treatment (+0.984 mass increment on the MS/MS spectra of de-glycosylated peptides).Exact composition and relative abondance of each precursor were estimated using Byologic Protein Metric software (see experimental section) and manually confirmed.

Identified peptide sequence Chain
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Figure S4 .
Figure S4.Identification of the band at 50,000 from SDS-PAGE of HSulf-2 treated by chondroitinase ABC.Sequence coverage (%) was determined after in gel trypsin treatment and nanoLC-ESI-MS/MS analysis.
Figure S15.Uncropped, full-length SDS-PAGE gels corresponding to (a) the SDS-PAGE analysis shown in figure 6A and (b) the SDS-PAGE analysis shown in figure 6C., detection with the SNAP-Vista Green.

Table S1 .
Putative sequences extracted from MALDI-TOF MS analyses of oligosaccharides issued from chondroitinase ABC depolymerization of the HSulf-2-bound GAG.Sequences in italics represent the least abundant reported in the whole literature.